Facilitation by intracellular carbonic anhydrase of Na+-HCO3- co-transport but not Na+/H+ exchange activity in the mammalian ventricular myocyte

Key points Carbonic anhydrase enzymes are said to enhance membrane H+ and HCO3- transport, an idea tested almost exclusively in heterologous cell-expression systems. In ventricular myocytes, inhibiting cytoplasmic enzyme activity slows membrane acid extrusion (HCO3- influx) on Na+-HCO3- co-transporters, with no effect on Na+/H+ exchangers. Inhibiting exofacial enzyme activity has no effect on either transporter. Mathematical modelling simulates the influence of carbonic anhydrase on Na+-HCO3- co-transport, provided the enzyme catalytically delivers H+ ions to the transporter (across a cytoplasmic nanodomain that is poorly accessible to intrinsic buffers), rather than catalysing protonation of the imported bicarbonate. Intrinsic cytoplasmic mobile buffers appear to deliver H+ to Na+/H+ exchangers, thus obviating the need for carbonic anhydrase. Conclusion: in native cardiac cells, intracellular carbonic anhydrase molecules partner Na+-HCO3- co-transporters but not Na+/H+ exchangers, to enhance flux activity. The enzyme thus plays a key role in supporting bicarbonate transport and pH control in the heart. Abstract Carbonic anhydrase enzymes (CAs) catalyse the reversible hydration of CO2 to H+ and HCO3- ions. This catalysis is proposed to be harnessed by acid/base transporters, to facilitate their transmembrane flux activity, either through direct protein-protein binding (a transport metabolon') or local functional interaction. Flux facilitation has previously been investigated by heterologous co-expression of relevant proteins in host cell lines/oocytes. Here, we examine the influence of intrinsic CA activity on membrane HCO3- or H+ transport via the native acid-extruding proteins, Na+-HCO3- cotransport (NBC) and Na+/H+ exchange (NHE), expressed in enzymically isolated mammalian ventricular myocytes. Effects of intracellular and extracellular (exofacial) CA (CA(i) and CA(e)) are distinguished using membrane-permeant and -impermeant pharmacological CA inhibitors, while measuring transporter activity in the intact cell using pH and Na+ fluorophores. We find that NBC, but not NHE flux is enhanced by catalytic CA activity, with facilitation being confined to CA(i) activity alone. Results are quantitatively consistent with a model where CA(i) catalyses local H+ ion delivery to the NBC protein, assisting the subsequent (uncatalysed) protonation and removal of imported HCO3- ions. In well-superfused myocytes, exofacial CA activity is superfluous, most likely because extracellular CO2/HCO3- buffer is clamped at equilibrium. The CA(i) insensitivity of NHE flux suggests that, in the native cell, intrinsic mobile buffer-shuttles supply sufficient intracellular H+ ions to this transporter, while intrinsic buffer access to NBC proteins is restricted. Our results demonstrate a selective CA facilitation of acid/base transporters in the ventricular myocyte, implying a specific role for the intracellular enzyme in HCO3- transport, and hence pH(i) regulation in the heart.